Elimination of misfolded proteins by ER-associated protein degradation (ERAD) ensures that proteins entering the secretory pathway are correctly folded and that ER stress is maintained at acceptable low levels. All variations of ERAD includes a protein translocation process termed retrotranslocation, in which ubiquitinated ERAD subsrates are selectively extracted by dedicated machinery from the ER before degradation by the cytosolic 26S proteasome. The overall goal of this project is to unravel the unknown mechanism of retrotranslocation. In the course of discovering the conserved HRD ERAD pathway, our laboratory has developed a variety of unique tools, techniques and expertise that will be brought to bear on the pressing and open question of retrotranslocation. Specifically, I will 1) perform biochemical and proteomic analyses on the prototypical ERAD substrate HMG-CoA reductase to understand the mechanism of its retrotranslocation from the ER and identify the key proteins involved in this process. Furthermore, I will 2) employ a genetic approach as an independent and complementary approach to identify retrotranslocation factors. They will include running traditional and array-based screens, combined with validation of candidate genes using direct in vivo and in vitro assays. Finally, 3) I will discern the roles of all discoveed factors in each known branch of ERAD. Taken together, these studies will reveal a key, universal and conserved process at the heart of ERAD involved in managing cell stress and a variety of clinical maladies. My studies in yeast will provide a new fundamental knowledge of the retrotranslocation process of ERAD. ERAD has been implicated in diseases such as aging, Alzheimer's disease, Huntington's disease, Parkinson's disease and in normal sterol regulation, my studies are pertinent for understanding how defects in ERAD are associated with these processes and maladies.